The present invention relates generally to isolated nucleic acids and the creation of vectors incorporating the same for the study and expression of genes in actinomycetes. The invention more particularly relates to novel genes isolated from a Micromonospora plasmid which can be used in the construction of vectors for the study and expression of genes and manipulation of metabolic pathways in actinomycete hosts.
Actinomycetes are branched filamentous Gram-positive bacteria. Streptomyces, Micromonospora, Nocardia, Actinoplanes, Saccharopolyspora, Actinomadura, Thermomonospora, Microbispora, Streptosporangium and others all represent genera of the Actinomycetes (Atlas of Actinomycetes, Asakura Publishing Co., Ltd 1996). Actinomycetes are very important industrially because they produce a variety of secondary metabolites such as antibiotics, herbicides, anticancer agents, antihelmintics, and anabolic agents (Demain., Appl. Microbiol and Biotechnology., 1999, 52:455-463). Antibiotics are a large and complex group of chemical substances which exert deleterious effects on other organisms, many of which organisms are harmful to humans. Thus, antibiotics are particularly important secondary metabolites to study and produce. This is especially true because many pathogens can develop antibiotic resistance to known antibiotics.
Given the actinomycetes"" proclivity for producing secondary metabolites such as antibiotics, it is especially advantageous to develop new tools such as vectors, promoters and the like to allow actinomycetes to be easily genetically manipulated. These tools would make it possible to control the levels of expression of genes encoding for secondary metabolites and also would make it possible to prepare derivatives or intermediates of these metabolites. In addition, the development of new vectors utilizing novel genes would make it possible to program microorganisms such as actinomycetes to produce recombinant products such as hybrid antibiotics via genetic engineering techniques.
In particular, it would be useful to construct replication, Escherichia coli-actinomycete shuttle, conjugal and integrating vectors which can be used to express genes in actinomycetes, for complementation experiments and heterologous gene expression.
Integrating vectors can be created which allow efficient integration of genes into a transformed host""s chromosome rather than replicating autonomously. They are particularly useful in transforming actinomycetes because of their high transformation rates, site specific integrative capacity and stable maintenance in host chromosomes without antibiotic selection. Vectors have been developed for use in actinomycetes that contain att/int functions for site specific integration of plasmid DNA. The two systems available make use of the att/int functions of bacteriophage phiC31 (U.S. Pat. No. 5,190,870) and plasmid pSAM2 (U.S. Pat. No. 5,741,675). However, there is a need for additional vectors with att/int functions for site-specific integration in M. carbonacea and similar organisms.
E. coli-actinomycete shuttle vectors can be created which will allow many traditional and facile molecular and genetic manipulations to be performed in E. coli. Plasmids developed in E. coli can then be introduced into actinomycetes to study their effects.
The ability to develop actinomycete conjugation vectors would allow intramycelial transfer which enables the transfer of plasmids between different phylogenetic classes of actinomycetes.
The present inventors have responded to the above needs and have isolated plasmid genes from Micromonospora rosaria, a species of actinomycete, in order to create vectors which can be used to express actinomycete genes, manipulate secondary metabolic pathways and create new metabolic products such as hybrid antibiotics. In addition, the isolated genes can be used to create replicating, E. coli-actinomycete shuttle, integrating, and intermycelial and intramycelial conjugation vectors for use in actinomycetes.
The present invention advantageously provides the polynucleotide sequences for the genes encoding the proteins and DNA regions required for the replication, transfer and integration of the M. rosaria pMR2 plasmid. As a result, the present invention provides the information needed to construct novel replicating, shuttle, integrating and conjugation vectors derived from the M. rosaria pMR2 plasmid. In addition, the invention provides hosts transformed with these novel vectors.
In one embodiment, the present invention provides isolated polynucleotides comprising sequences which are at least about ninety percent homologous to the nucleotide sequences set forth in SEQ ID NOS: 2-15. In a preferred embodiment, the present invention provides isolated polynucleotides comprising sequences set forth in SEQ ID NOS: 2-15. These isolated polynucleotides encode novel genes and DNA sequences involved in plasmid replication, integration, excision, and intermycelial and intramycelial conjugation. In preferred embodiments, these isolated sequences encode a site-specific integrase, an excisionase, an attachment site for plasmid integration, inter- and intramycelial transfer genes, origin of replication, and plasmid regulatory proteins.
In another embodiment, the present invention provides recombinant vectors comprising a sequence having at least about ninety percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2-15. In a preferred embodiment, the invention provides recombinant vectors comprising one or more of SEQ ID NOS: 2-15. In an especially preferred embodiment, the recombinant vector is an integration vector capable of integrating into the chromosome of the host cell. In yet another preferred embodiment, the recombinant vector is a vector capable of inter- and intramycelial conjugation in actinomycetes. In still another preferred embodiment, the recombinant vector is capable of replicating in both E. coli and actinomycete species.
In yet another embodiment, the present invention provides host cells comprising the vectors of the instant invention. In a preferred embodiment, the host cells are bacterial. In an especially preferred embodiment, the host cells are Micromonospora carbonacea, Micromonospora halophitica, and/or Streptomyces lividans. 
In a final embodiment, the present invention provides an isolated polynucleotide comprising a sequence having at least about ninety percent homology to the sequence set forth in SEQ ID NO:1. Preferably, the polynucleotide comprises a sequence identical or nearly identical to the sequence set forth in SEQ ID NO: 1.
These and other aspects of the invention are better understood by reference to the following Detailed Description and Examples.